1. Field of the Invention
The invention relates to a method for the detection of incorrect deposition on a MALDI sample support with several separate sample sites. The invention furthermore relates to methods for the mass-spectrometric detection of analyte molecules with integrated detection of contamination on the MALDI sample support.
2. Description of the Related Art
Matrix-assisted laser desorption/ionization (MALDI) is a method of ionizing molecules. Since first being developed in the 1980s, it has become more and more important for the mass spectrometry of large molecules and polymers as well as biopolymers, such as proteins.
MALDI is based on the simultaneous crystallization of a matrix substance and an analyte substance with a 100- to 100,000-fold molar excess of matrix molecules. Analyte molecules are embedded into the crystals of the MALDI matrix as it forms. Successful, simultaneous crystallization typically requires a matrix/analyte ratio of around 104 to 1. Small organic molecules which strongly absorb energy at the laser wavelength used, for example nitrogen lasers at a wavelength of 337 nm, are regularly selected as matrix substances. Examples are sinapic acid, 2,5-dihydroxybenzoic acid or α-cyano-4-hydroxycinnamic acid. The excitation is performed using short, high-energy laser pulses, of two to five nanoseconds pulse duration, for example. After relaxation in the crystal lattice, the excitation leads to explosive particle detachments at the surface of the crystal. The embedded analyte molecules are thus released together with the matrix and ionized. The ionized analyte molecules are thus converted into the gaseous phase and can then be transferred into the vacuum of a mass spectrometer and analyzed mass-spectrometrically.
The important aspects for a mass-spectrometric measurement in which the ions are produced by MALDI are the type of sample preparation and the method of applying of the samples onto the sample support, which is often made of metal, or occasionally of semiconductor material or electrically conductive plastic. There are various ways to apply the sample, such as the dried droplet method or thin layer preparation, which are all known in the Prior Art and shall not be dealt with further here.
MALDI samples are usually prepared on flat sample supports with a specific number of separate sample sites, which are typically arranged in a grid. The number can vary from 96 to 384 to 1536 sample sites, for example, depending on the design of the sample support. Deposition errors can occur when a sample is being prepared on such a sample site, especially when the matrix solution is being applied. It is possible, for example, for matrix solution to overflow from one sample site to another sample site that is not actually intended to be spotted because the volume of matrix solution exceeds the capacity of the targeted sample site. With automated dispenser units, in particular, there is an additional risk that a dispensed liquid volume is not deposited accurately onto a sample site but away from its center, onto the sample support, because the positioning device is inexact. Incorrect deposition can also be caused by a dispenser capillary releasing a volume of liquid not along the axis of the capillary but at an angle to it if, for example, the capillary tip has become partially clogged, and is thus geometrically constricted in an unpredictable way.
All the above-mentioned causes of incorrect deposition may result in a sample not being prepared on the intended site on the sample support, but outside it, which increases the risk of cross-contamination. In all cases it is highly probable that the area on the sample support between the individual sample sites, which should really remain free of sample and/or matrix substance and thus of contamination, is affected by the incorrect depositions.
The Prior Art, for example the patent publications EP 1 763 061 A2 and US 2002/0191864 A1, discloses that when sample preparation has been completed, a sample support is examined by means of a camera and connected image analysis unit to detect the presence and location of applied samples, as explained in Paragraph 0056 of the European patent application, for example. FIG. 4 of the US American patent application shows, by way of example, what MALDI depositions can look like. The prepared sample identified with the reference labels 16b and 16e, in particular, must be considered as critical because they are simultaneously close to two adjacent sample sites and can thus falsify the assignment of sample to sample site on the MALDI sample support. The sample with the reference label 16c is also fundamentally unfavorable because a large part of the sample volume is outside the area of the site which is intended for the sample application. This means that it can take a long time until the desorption laser, which is scanning the sample site, is directed at a point on the sample support which is spotted with a sample and is thus productive. It can also be the case that not enough productive points are bombarded and therefore the mass-spectrometric signal recorded has a high proportion of noise.
In order to implement the principles of the ideas explained in the documents listed above, optical images of the spotted sample supports must be taken and evaluated, which significantly increases the procedural effort for a MALDI ionization. There is therefore a need to specify alternative methods for the detection of incorrect depositions or contamination on MALDI sample supports which require less time and effort and can, in particular, be carried out with the available instruments, comprising a MALDI ion source and a connected mass spectrometer or mass analyzer.